1. Field of the Invention
The present invention relates to a method for the detection and/or determination of nucleic acid in which the nucleic acid is hybridized to a labelled oligonucleotide, comprising a nucleic acid sequence complementary to at least part of the nucleic acid to be detected and/or determined, thereby forming a duplex nucleic acid which is separated from unhybridized labelled oligonucleotide by gel electrophoresis prior to detection of the labelled duplex nucleic acid in a gel.
2. Description of the Prior Art
Kumar et al. have described a method, the "probe shift assay" for the detection of PCR amplified DNA sequences In one of their publications, Aids Research and Human Retroviruses, Vol. 5, No. 3, 345-353, 1989, an assay is described in which HIV sequences were detected in PCR amplified DNA samples by adding a .sup.32 P-radioactive labelled oligonucleotide to a sample containing amplified DNA, and hybridizing the oligonucleotide to the amplified and denatured DNA. After the radioactive labelled oligonucleotide and amplified DNA were annealed, the heteroduplex formed was separated from the free radioactive labelled oiigonucleotide by gel electrophoresis on a non denaturing polyacrylamide gel. Following gel electrophoresis an X-ray film was exposed to the gel.
One disadvantage of such a system is that long exposure times -up to several hours- are required for detection of the radio labelled oligonucleotide in the gel. Another disadvantage of this system is that the use of radioactive material requires that these assays be performed by specially trained personnel in laboratories well adapted for the use of radioactive material.
In another article of Kumar et al., Technique, Vol. 2, No. 2, 101-108, 1990, an assay is described, based on the same principle, in which fluorescent labelled oligonucleotides were used instead of the previously described radioactive oligonucleotides. A 32-mer oligonucleotide was modified during synthesis by the attachment of fluorescein molecules to T residues. The label can now be detected in the gel by laser excitation. This method however requires the use of complicated laser instrumentation and is therefore not generally suitable for diagnostic purposes.